Overview
Our long-range goals are to understand epigenetic regulation of gene expression in mammalian development and disease. An important question is to understand the different epigenetic conformations that distinguish differentiated cell states and to define strategies to transdifferentiate one differentiated cell type into another. Embryonic stem cells are of major significance because they have the potential to generate any cell type in the body and, therefore, are of great interest for regenerative medicine. A major focus of our work is to understand the molecular mechanisms that allow the reprogramming of somatic cells to an embryonic pluripotent state and to use the potential of patient specific pluripotent cells to study complex human diseases.

Research Summary

Embryonic stem cells and the control of self-renewal:
The transcription factors Oct4, Sox2, and Nanog have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have, in collaboration with the Young lab, identified Oct4, Sox2, and Nanog target genes using genome-scale location analysis. We found, surprisingly, that Oct4, Sox2, and Nanog co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also show that Oct4, Sox2, and Nanog collaborate to form regulatory circuitry in ES cells consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how Oct4, Sox2, and Nanog contribute to pluripotency and self-renewal.

Nuclear Cloning and the Reprogramming of the Genome:
A major issue raised some 50 years ago in seminal frog cloning experiments was the question of whether nuclei of terminally differentiated cells can be reprogrammed to generate animals after nuclear transfer. We have shown that cloned mice can be derived from mature B and T cells or from mature neurons by nuclear transfer, demonstrating that the genome of terminally differentiated cells can be reprogrammed to direct development of a new animal.
Transplantation of functional cells for tissue repair represents an exciting strategy for possible treatment of patients suffering from degenerative ailments such as Diabetes, Parkinson’s or heart disease. A major impediment is the availability of suitable cells that could be used for transplantation and would not be rejected due to immunological incompatibility. The promise of nuclear transplantation (sometimes called “therapeutic cloning”) is to provide “customized” embryonic stem cells that could be used for patient specific therapy. We have performed a "proof of principle" experiment in a mouse disease model to provide evidence that therapeutic cloning combined with gene therapy represents a valid strategy for transplantation therapy. However, the complexity and inefficiency of the NT procedure makes it unlikely that “therapeutic cloning” could be used in a routine clinical setting in the foreseeable future.

In vitro reprogramming of somatic cells to a pluripotent state:
The recent success in reprogramming somatic cells to pluripotent Induced Pluripotent Stem (iPS) cells by defined factors has opened exciting possibilities not only for the investigation of complex human diseases in the Petri dish but also for the ultimate application in transplantation therapy. A major focus of our work is (i) to study the molecular mechanisms of somatic reprogramming and to devise efficient approaches for the reprogramming of mouse and human somatic cells; (ii) to derive patient specific iPS cells for the generation of tissue culture models of major human diseases; and (iii) to establish proof of principle experiments for the eventual therapeutic use of iPS cells.

(i) Molecular mechanisms: The study of induced pluripotency is complicated by the need for infection with high titer retroviral vectors resulting in genetically heterogeneous cell populations. We have generated genetically homogeneous “secondary” mouse and human somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. This system facilitates the characterization of reprogramming and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors. For example, the secondary system has allowed us to define the role of stochastic epigenetic events and of cell proliferation during the reprogramming process. Also, we have been able to generate iPS cells and mice from different somatic donor cells such as mature B cells, intestinal cells and neural precursors.

(ii) Patient specific iPS cells: Most current methods for reprogramming human somatic cells preclude consideration for cell replacement therapies since they rely on the delivery of the four reprogramming factors by retroviral transduction, which carries the risk of tumor formation. Also, a concern is that the low level of provirus expression that is consistently detected in the iPS cells may affect other biological characteristics such as differentiation potential. An important issue of the field is to generate vector free iPS cells.
To decrease the possibility of provirus-mediated insertional mutagenesis we have generated human iPS cells with a single-copy proviral insert by using a polycistronic vector to deliver the reprogramming factors. In addition we have shown that fibroblasts from patients with sporadic Parkinson’s Disease (PD) can be efficiently reprogrammed using vectors that could be excised by Cre-mediated deletion thus generating Parkinson patient-derived iPS cells that are free of the reprogramming factors. The cells maintained all of the characteristics of a pluripotent ES cell-like state after removal of the transgenes. Importantly, genome wide gene expression analysis revealed that the factor-free iPS cells clustered more closely with embryo-derived human ES cells than with the parental virus-carrying iPS cells, consistent with the notion that the presence of vectors may affect the properties of iPS cells. A major goal is to establish in vitro differentiation systems that allow us to study the pathogenesis of neurodegenerative diseases such as Parkinson’s, Alzheimer disease or ALS in the Petri dish and to eventually isolate small molecules that could be used for therapy.

(iii) Therapeutic potential of iPS cells: One of the most exciting applications of the iPS cell technology is the use of patient specific cells for the treatment of diseases such as Diabetes, Parkinson’s or blood disorders. We have, as a proof of principle therapy study, demonstrated that iPS cells derived from autologous skin cells of a mouse with Sickle Cell Anemia can induce complete recovery when transplanted into the mutant mice. In a second model we demonstrated the integration of iPS derived neurons into fetal brain and the subsequent reduction of symptoms in rats with Parkinson’s disease. Both of these models are encouraging and argue that iPS cells can be used for the therapy of major diseases.

(iv) Genetic manipulation of human ES and iPS cells: In contrast to mouse ES cells, homologous recombination in human pluriptient cells is extremely inefficient impeding progress in using the potential of human ES and iPS cells for disease research. We have used Zinc Finger Nucleases (ZFN) and TALENs (Transcription Activator Effector Like Nucleases) to engineer genes. These novel approaches are efficient in introducing specific alterations into cellular genes including deletions, insertions and point mutations. It is likely that these approaches will vastly expand our ability to establish in vitro model system to study complex human disease in the Petri dish by generating genetically defined disease specific and matched control iPS or ES cells.

(v) Generation of human ES cells with properties of mouse ES cells: A major impediment for realizing the potential of human ES cells for the study of diseases is the difficulty to grow and to genetically modify the cells. Thus, in contrast to mouse ES cells, human ES cells have a low single cell cloning efficiency, depend on TGFb and activin instead of LIF/STAT3 for self-renewal, are very inefficient in homologous recombination impeding the generation of gene targeting and have to be passaged mechanically instead of by trypsin to avoid chromosomal aberrations. In addition, female mouse ES cells are pre-inactivation with both X chromosomes being active (XaXa) whereas conventional human ES cells have already undergone X inactivation (XiXa). We have succeeded in converting conventional human ES cells into a pluripotent state that resembles mouse ES cells by all the criteria mentioned above. It is hoped that these new human ES cells will overcome the many obstacles that presently impede the use of human ES cells for disease research.

Cancer:
The involvement of DNA methylation in cancer has been controversial: both hypomethylation as well as hypermethylation have been associated with malignant transformation. When the MTase mutation was introduced into mice with a genetic predisposition to colon cancer, a surprising result was seen: the MTase enzyme level directly correlated with the development of cancer. This argued that the MTase enzyme itself may act as an oncogenic determinant and may be a potentially attractive drug target for cancer prevention and treatment.

Genomic hypomethylation is a widely observed and early step in human tumorigenesis. Using different mutant alleles of the Dnmt1 gene we have shown that hypomethylation results in a substantial increase in the genomic mutation rates, the mutations being caused by enhanced mitotic recombination. These results are significant as they may explain the selective advantage of hypomethylation in early stages of transformation: hypomethylation leading to genomic instability may provide the incipient tumor cell with a mechanism to efficiently delete tumor suppressor genes by LOH. The MTases Dnmt3a and b are the best candidates to cause the silencing of tumor suppressor genes by de novo methylation. Indeed, our results show that Dnmt3b is crucially important for intestinal cancer as it promotes the silencing of tumor suppressor genes. Surprisingly recent results show that Dnmt3a instead of Dnmt3b plays a key role in lung cancer.

The importance of epigenetic alterations leading to cancer was demonstrated by the reversal of the malignant state. Nuclear transplantation was used to reprogram cancer cells to pluripotent ES cells that were able to generate chimeric mice consistent with the notion that the malignant phenotype of the donor cancer cells was largely determined by epigenetic, i.e. reversible alterations. Current efforts are directed towards using in vitro reprogramming to revert the phenotype of tumor cells including cancer stem cells.

Selected Publications

Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956.

Hanna, J., Cheng, A.W., Saha, K., Kim, J., Lengner, C.J., Soldner, F., Cassady, J.P., Muffat, J., Carey, B.W., and Jaenisch, R. (2010). Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci U S A 107, 9222-9227.

Hanna, J., Markoulaki, S., Mitalipova, M., Cheng, A.W., Cassady, J.P., Staerk, J., Carey, B.W., Lengner, C.J., Foreman, R., Love, J., et al. (2009a). Metastable Pluripotent States in NOD-Mouse-Derived ESCs. Cell Stem Cell 4, 513-524.

Hanna, J., Markoulaki, S., Schorderet, P., Carey, B.W., Beard, C., Wernig, M., Creyghton, M.P., Steine, E.J., Cassady, J.P., Foreman, R., et al. (2008b). Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250-264.

Hanna, J., Saha, K., Pando, B., van Zon, J., Lengner, C.J., Creyghton, M.P., van Oudenaarden, A., and Jaenisch, R. (2009b). Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595-601.

Hanna, J., Wernig, M., Markoulaki, S., Sun, C.W., Meissner, A., Cassady, J.P., Beard, C., Brambrink, T., Wu, L.C., Townes, T.M., et al. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920-1923.

Hochedlinger, K., and Jaenisch, R. (2002). Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 415, 1035-1038.

Lengner, C.J., Gimelbrant, A.A., Erwin, J.A., Cheng, A.W., Guenther, M.G., Welstead, G.G., Alagappan, R., Frampton, G.M., Xu, P., Muffat, J., et al. (2010). Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 141, 872-883.

Rideout, W.M., 3rd, Hochedlinger, K., Kyba, M., Daley, G.Q., and Jaenisch, R. (2002). Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109, 17-27.

Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G., Cook, E., Hargus, G., A., B., Cooper, O., Mitalipova, M., et al. (2009). Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964-977.

Wernig, M., Lengner, C.J., Hanna, J., Lodato, M.A., Steine, E., Foreman, R., Staerk, J., Markoulaki, S., and Jaenisch, R. (2008). A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 26, 916-924.

Wernig, M., Meissner, A., Cassady, J., and Jaenisch, R. (2008). C-Myc Is Dispensable for Direct Reprogramming of Mouse Fibroblasts. Cell Stem Cell 2, 10-12.

Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324.

Wernig, M., Zhao, J.P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Constantine-Paton, M., Isacson, O., and Jaenisch, R. (2008). Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci U S A 105, 5856-5861.

Soldner, F, Laganiere J, Cheng AW, Hockemeyer D, Gao Q, Alaappan R, Khurana V, Golbe Ll, Myers RH, Lindquist S, Zhang L, Guschin D, Fong LK, Vu BJ, Meng X Urnov FD, Rebar EJ, Gregory PD, Zhang HS, & Jaenisch R. (2011) Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318-31.

Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD & Jaenisch R. (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731-4.

Kim JP, Su SC, Wang H, Cheng AW, Cassady JP, Lodato MA, Lengner CJ, Chung C-Y, Dawlaty MM, Tsai L-H & Jaenisch R. Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell (in press).

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